Abstract
The histoarchitecture and function of eye and forebrain depend on a well-controlled balance between cell proliferation and differentiation. For example, the binding of the cell cycle regulator GEMININ to CDT1, which is a part of the pre-replication complex, promotes cell differentiation. Homeodomain transcription factors SIX3 and SIX6 also interact with GEMININ of which SIX3-GEMININ interaction promotes cell proliferation, whereas the nature of SIX6-GEMININ interaction has not been studied to date. We investigated SIX3/SIX6 and GEMININ interactions using bimolecular fluorescence complementation, surface plasmon resonance and isothermal titration calorimetry. Interactions between SIX3/SIX6 and GEMININ were detected in mammalian cells in culture. The presence of the C-terminal regions of SIX3 and SIX6 proteins, but not their SIX domains or homeodomains as previously thought, were required for interaction with GEMININ. Interestingly, the disordered C- and N- terminal regions of GEMININ were involved in binding to SIX3/SIX6. The coiled-coil region of GEMININ, which is the known protein-binding domain and also interacts with CDT1, was not involved in GEMININ-SIX3/SIX6 interaction. Using SPR and ITC, SIX3 bound GEMININ with a micromolar affinity and the binding stoichiometry was 1:2 (SIX3 - GEMININ). The present study gives new insights into the binding properties of SIX proteins, especially the role of their variable and disordered C-terminal regions.
Highlights
The homeodomain-containing transcription factors SIX3 and SIX6 play a crucial role in eye and forebrain development in vertebrates [1,2,3]
The homeodomain-containing transcription factors SIX3 and SIX6 play important roles in eye and forebrain development. In understanding their roles in development, attention has been largely focused on the two evolutionary conserved domains: the homeodomain (DNAbinding domain) and the SIX domain, previous studies suggested possible functions in phosphorylation and DNA binding for the non-conserved C-terminal regions [15,28]
Using bimolecular fluorescence complementation (BiFC) we investigated whether the interactions occur between GEMININ and SIX3/SIX6 testing homeodomains (HDs), SIX domains (SDs) or C-terminal regions of the SIX3/SIX6 proteins
Summary
The homeodomain-containing transcription factors SIX3 and SIX6 play a crucial role in eye and forebrain development in vertebrates [1,2,3]. They influence the expression of various proteins through the direct binding to their regulatory DNA sequences as well as through the interaction with other proteins (co-factors) [4], for example, the DNA replication inhibitor GEMININ [5]. GEMININ itself inhibits cell cycle progression by binding to CDT1 (part of the pre-replication complex) [6,7,8,9] and by controlling CDT1 levels [10].
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